The laser imaging system of the present invention is designed to provide a means for rapid quantitative image capture and digitization where requirements for field size, speed, spatial resolution, dynamic range, and low light sensitivity are not adequately provided for by conventional optical imaging devices.
Specifically, optical devices that incorporate lenses to form a real or virtual image at any point in the system are limited with respect to the target field size, given a particular effective numerical aperture (NA) and spatial resolution. For instance, a microscope provides a means of imaging using lenses of high light gathering power, i.e., high numerical aperture (NA). Typically the value of the NA for an objective lens with 20X is 0.50 (range 0.40-0.75). However, this type of lens is capable of imaging a field of only 3 square millimeters. See for example FIG. 1. If a field of 20 mm.times.40 mm is to be imaged (approximately the area of a standard microscope slide), then the maximum effective NA for conventional optical lenses would be approximately 0.04-0.10. For this reason highly sensitive fluorescence measuring devices have incorporated microscopes with high NA lenses equipped with mechanical stages to move the target and effect the wide field scan.
Gross mechanical translation of targets such as microscope slides is generally slow and subject to maintenance problems related to wear and failure. The ability of mechanical stages to physically move the target is limited by the need for high positioning accuracy which is typically within 0.5 um. Thus, a high precision autostage will have a maximal translation rate of only about 20 mm/second. Other additional complicating factors involved in image capture require that the mechanical stage stop at each field location long enough for the capture device to electronically transfer the image data to some storage form. Thus, these conventional optical devices, when used for high resolution, low light level scans (e.g., immunofluorescence), can require several hours to capture target fields the size of a standard microscope slide.
If a field of 20 mm.times.40 mm is to be imaged, then compromises are necessary when using optical lenses. Wide field imaging with effective numerical apertures of 0.9 or higher has led to complex designs and instruments that require hours for image capture. A significant improvement upon these conventional optical devices is the flying spot scanner. However, the flying spot scanners available today including the most recent laser-induced devices, have a significant problem, which is the simple fact that the focal point of the light beam is fixed. Therefore, for a two dimensional scan, the beam spot is found on a curved surface. Correction for this curvature in the present day flying spot scanner requires the incorporation of cumbersome multisided spinning mirrors of complex design. These mirrors afford little control over the location of the laser beam. Conventional flying spot scanners also move the laser in a preprogrammed ballistic direction. The direction and velocity parameters are preprogrammed and the scan cannot be controlled outside of the programming.
Another problem associated with the design of a flying spot scanner for use in fluorescence detection or forward scatter detection is the relatively low efficiency light gathering available using conventional design which use lenses for detection. For instance, a design described by Slomba, et al., (J. Assoc. Adv. Medical Instr., 6:230, 1972) for a flying spot fluorescence detection is capable of capturing less than 1% of the total target emission. This is equivalent to an unacceptable NA of 0.10.
Imaging systems can be designed for a large variety of applications. Generally, however, the requirements of target field size, NA, and spatial resolution dictate specific structures for specific applications. There is little crossover in design for the varying applications. The imaging system of the present invention overcomes the design limitations of current imaging devices and is easily adaptable to perform a large number of imaging operations.